PROJECT SUMMARY/ABSTRACT The overall goal of the present work is to determine the mechanisms that permit the emergence of B lymphoid cells in the extraembryonic yolk sac and the para aortic splanchnopleura (P-Sp) and facilitate the homing of these cells into the peritoneal cavity. Though the extraembryonic yolk sac is the first site of primitive red blood cell, megakaryocyte, and macrophage production, it is controversial whether it is a site of B lymphoid emergence. An intraembryonic site, the aorta-gonad-mesonephros (AGM) region derived from the P-Sp, is currently recognized as the first site of production of HSCs capable of repopulating the hematopoietic compartment of lethally irradiated adult mice and is reported to possess the potential to give rise to B lymphoid cells upon in vitro co-culture with a certain stromal cell line. However, after the systemic circulation has commenced, it remains unclear and unproven whether the B cell progenitors are moving from the yolk sac into the P-Sp or vice versa. Indeed, the most formidable obstacle encountered when attempting to define the onset of B lymphopoiesis in the developing murine embryo, is the onset of cardiac contractions and establishment of the circulation. We have developed a novel approach that permits determination of exactly where and when hematopoietic cells emerge in the yolk sac and P-Sp. Ncx1 encodes a sodium-calcium exchange protein that is expressed only in cardiomyocytes through embryonic day (E) 11.0. Embryos lacking Ncx1 are unable to initiate cardiac contractions preventing the onset of the systemic circulation. Lack of blood flow is initially tolerated without evidence of maldevelopment, however embryos demonstrate growth retardation by E10.0 and die by E11.0. Using Ncx1 mutant and wild type (WT) mice, we have recently reported that all hematopoietic definitive progenitor cells that seed the E 10.0 fetal liver are yolk sac-derived. We have also reported that the potential for forming B lymphoid cells is present in the E 9.5 yolk sac and P-Sp of wild type and Ncx1 null mice; confirming that B lymphoid cells arise autonomously in the yolk sac and P-Sp. A host of questions now emerge that we address in the following aims: Aim 1: To delineate the temporal and spatial emergence of B lymphoid cells in the yolk sac and P-Sp and to define B cell specification to B1 and/or B2 lymphoid lineages from the emergent cells in these sites. Aim 2: To define the growth factor signaling pathways required for yolk sac and P-Sp B lymphopoiesis. Aim 3: Define the molecular pathway for yolk sac and P-Sp derived B cell homing to the peritoneal cavity. These studies will define the mechanism of B lymphocyte emergence, determine whether differences in B cell specification are intrinsic and restricted, and may clarify the origin of the B1 lymphocytes that secrete natural antibodies; a population of cells important in human development of autoimmune diseases and leukemia.